Labeling of a protein, cell, or organism of interest plays a prominent role in many biochemical, molecular biological and medical diagnostic applications. A variety of different labels have been developed and used in the art, including radiolabels, chromolabels, fluorescent labels, chemiluminescent labels, and the like, with varying properties and optimal uses. However, there is continued interest in the development of new labels. Of particular interest is the development of new protein labels, including fluorescent protein labels.
Green Fluorescent Protein (GFP), its mutants and homologs are widely known today due to their intensive use as in vivo fluorescent markers in biomedical sciences discussed in detail by Lippincott-Schwartz and Patterson in Science (2003) 300(5616):87-91). The GFP from hydromedusa Aequorea aequorea (synonym A. victoria), discovered by Johnson et al. in J Cell Comp Physiol. (1962), 60:85-104, was found as a part of bioluminescent system of the jellyfish where GFP played role of a secondary emitter transforming blue light from photoprotein aequorin into green light. Then, similar proteins were isolated from several bioluminescent coelenterates including hydroid medusa Phialidium gregarium, sea pansy Renilla (class Anthozoa) and others (see Ward et al. in Photochem. Photobiol. (1982), 35: 803-808; Levine et al. in Comp. Biochem. Physiol. (1982), 72B: 77-85; Chalfie in Photochem. Photobiol. (1995), 62:651-656). All these proteins display green fluorescent (emission at 497-509 nm) and functioned as the secondary emitters in bioluminescence. Fluorescent proteins were also isolated from Physalia species and their N-terminal amino acid sequences were determined (WO 03/017937).
cDNA encoding A. victoria GFP was cloned by Prasher et al. (Gene (1992), 111(2):229-33). It turned out, that this gene can be heterologically expressed in practically any organism due to unique ability of GFP to form fluorophore by itself (Chalfie et al., Gene (1992), 111(2):229-233). This finding opens broad perspectives for use of GFP in cell biology as a genetically encoded fluorescent label.
The GFP was applied for wide range of applications including the study of gene expression and protein localization (Chalfie et al., Science 263 (1994), 802-805, and Heim et al. in Proc. Nat. Acad. Sci. (1994), 91: 12501-12504), as a tool for visualizing subcellular organelles in cells (Rizzuto et al., Curr. Biology (1995), 5: 635-642), for the visualization of protein transport along the secretory pathway (Kaether and Gerdes, FEBS Letters (1995), 369: 267-271).
A great deal of research is being performed to improve the properties of GFP and to produce GFP reagents useful and optimized for a variety of research purposes. New versions of GFP have been developed, such as a “humanized” GFP DNA, the protein product of which has increased synthesis in mammalian cells (Haas, et al., Current Biology (1996), 6: 315-324; Yang, et al., Nucleic Acids Research (1996), 24: 4592-4593). One such humanized protein is “enhanced green fluorescent protein” (EGFP). Other mutations to GFP have resulted in blue-, cyan- and yellow-green light emitting versions. Despite the great utility of GFP, however, other fluorescent proteins with properties similar to or different from GFP would be useful in the art. In particular, benefits of novel fluorescent proteins include fluorescence resonance energy transfer (FRET) possibilities based on new spectra and better suitability for larger excitation. In 1999 GFP homologs were cloned from non-bioluminescent Anthozoa species (Matz et al., Nature Biotechnol. (1999), 17: 969-973). This discovery demonstrated that these proteins are not necessary component of bioluminescence machinery. Anthozoa-derived GFP-like proteins showed great spectral diversity including cyan, green, yellow, red fluorescent proteins and purple-blue non-fluorescent chromoproteins (CPs) (Matz et al., Bioessays (2002), 24(10):953-959).
The major drawback of the Anthozoa-derived GFP-like is strong oligomerization that hampers the use of these proteins in many applications (Lauf et al., FEBS Lett. (2001), 498: 11-15; Campbell et al., Proc. Natl. Acad. Sci. USA (2002), 99: 7877-7882; Mizuno et al., Biochemistry (2001), 40: 2502-2510). Accordingly, it is an object to provide novel monomeric fluorescent proteins of different colors as well as DNAs encoding them that do not suffer from the drawbacks of the known GFP.
Hydrozoa species are potential source of such proteins. Except Aequorea victoria GFP and GFP homologues from other Aequorea species, like very close GFP homologues from Aequorea macrodactyla (GenBank accession numbers AF435427-AF435433) and Aequorea coerulescens (Gurskaya et al., Biochem J. (2003), 373(Pt 2): 403-408), no other genes encoding fluorescent proteins from Hydrozoa are cloned to date although some of them were characterized at protein level very long ago. Cloning and mutagenesis of the non-Aequorea Hydrozoa fluorescent proteins is a perspective way to obtain novel fluorescent labels with improved features.